title: Blood and immune cell development in human fetal bone marrow and in Down syndrome

city: Cambridge

first: Bertie

last: Gottgens

address: Jeffrey Cheah Biomedical Centre

institute: University of Cambridge

summary: Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 11-12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. B-lymphocytes expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window.

overall_design:
Sample RBG33493 is a pool of MNCs from three stage 3 fetal bone marrow samples (F90, F103, F107). Sample RBG33494 is a pool of CD34+ cells from two adult bone marrow samples (CIMA-60, CIMA-58) and one stage 3 fetal bone marrow sample (F103). Sample RBG33496 is a pool of CD34+ cells from two adult bone marrow samples (CIMA-90, CIMA-100) and one stage 3 fetal bone marrow sample (F107). Sample RBG33500 is a pool of CD34+ cells from two stage 2 fetal liver samples (F94 and F96) and one stage 1 fetal liver sample (F109). Sample RBG33503 is CD34+ cells from a one stage 2 fetal bone marrow sample (F96). Samples RBG33507 and RBG33508 are technical replicates of a pool of CD34+ cells from three stage 3 fetal liver samples (F90, F103, F107). Sample RBG31066 is a pool of CD34+ cells from four new born cord blood samples (XN3392). Individuals not analysed for this article - CIMA-60, CIMA-58, CIMA-90, CIMA-100, F94 and F109.

Supplementary_files_format_and_content:
ADT_DSBnormalised_postmRNAoverlap_CD34Sinu.csv.gz: CD34+ sinusoidal EC DSB normalised counts.
overlap_ADTonmRNA_DSBnormalised_FBM-MNCs.csv.gz: FBM EC DSB normalised counts.
overlap_ADTonmRNA_raw_FBM-MNCs.csv.gz: RAW protein - Post overlap with  mRNA FBM MNCs.
overlap_ADTonmRNADSBnormalised_FL-FBM-CB.csv.gz: CD34+ progenitor EC DSB normalised counts.
overlap_ADTonmRNAraw_FL-FBM-CB.csv.gz: RAW Protein - Post QC + post overlap with demultiplexed mRNA Combination of FL-FBM-CB.
postQC_ADT_raw_FBM-MNCs.csv.gz: RAW protein - Post-QC FBM MNCs.
postQC_mRNAraw_FBM-MNCs.csv.gz: RAW mRNA - Post QC (+post doublet removal) FBM MNCs.
postQC_mRNAraw_FL-FBM-CB.csv.gz: RAW mRNA - Post QC (post doublet removal, post demultiplexing when needed) Combination of FL-FBM-CB.
preQC_ADTraw_FBM-MNCs.csv.gz: RAW protein - Post-CITE-seq-Count (pre-QC) FBM MNCs.
preQC_ADTraw_FL-FBM-CB.csv.gz: RAW Protein - Post-CITE-seq-Count (pre-QC) Combination of FL-FBM-CB.
preQC_mRNAraw_FBM-MNCs.csv.gz: RAW mRNA - Post-cell ranger (pre-QC) FBM MNCs.
preQC_mRNAraw_FL-FBM-CB.csv.gz: RAW mRNA - Post-cell ranger (pre-QC) Combination of FL-FBM-CB.
Raw_ADT_CD34Sinu_FLFBM.csv.gz: RAW Protein - Expression data.
Raw_mRNA_CD34Sinu_FLFBM.csv.gz: RAW mRNA - Sinusoidal endo RNA.

first: Rebecca

last: Hannah

first: Nicola

middle: K

last: Wilson

first: Mariana

last: Quiroga Londoño


title: ATACseq, CutnRun and RNAseq data from sensory progenitors and hair cells, from wild type and Pou4f3-mutant cochlea

status: Public on Jul 09 2021

city: Los Angeles

first: Neil

last: Segil

state: CA

address: 1425 san pablo st, BCC 503K

department: BCC

country: USA

institute: University of Southern California

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Hearing and balance are mediated in vertebrates by inner ear mechanosensory hair cells. Hair cell development, maturation and survival require POU4F3, a class IV POU domain transcription factor that is expressed in hair cells shortly after they start to differentiate. Here, we show that POU4F3 has pioneer factor activity, and is necessary to promote chromatin accessibility at approximately half of the distal regulatory elements that appear as hair cells differentiate. Pou4f3 expression in the inner ear is initiated by ATOH1, a bHLH transcription factor that is also necessary for hair cell differentiation and survival. We show that ATOH1 also binds a large number of POU4F3-dependent distal regulatory elements, suggesting a synergistic feed-forward model in which ATOH1 induces POU4F3, which then acts to promote ATOH1-dependent hair cell differentiation.

overall_design: In the present study, we used CUT&RUN (Skene and Henikoff, 2017) to identify the distal regulatory elements of differentiating hair cells that bind ATOH1. We used ATACseq (Buenrostro et al., 2013) to show that the majority of these ATOH1-bound distal elements fall within nucleosome-occupied, silent chromatin in sensory progenitors from which hair cells derive. We use RNAseq to show that POU4F3-dependent ATOH1 targets control the expression of deaf-related genes in the developing hair cells

first: Yu

last: Haoze

first: Tao

last: Litao

first: Segil

last: Neil


title: Expression data from malaria-specific mouse CD4 T cell (PbT-II) subset after Plasmodium berghei ANKA infection

status: Public on Jul 11 2021

city: Nagasaki-shi

first: Mbaya

middle: Ntita

last: Felly

state: Nagasaki

address: Sakamoto

department: Molecular Microbiology and Immunology

country: Japan

institute: Nagasaki University

summary: The multiple species of Plasmodium that infect humans are highly polymorphic and induce various infections ranging from asymptomatic state to severe life-threatening diseases such as cerebral malaria. However, how the differences between the parasites affect host immune responses during blood-stage infection remains largely unknown. In this study, we investigated the CD4+ T-cell immune responses in T-cell receptor-transgenic mice infected simultaneously with P. berghei ANKA (PbA) and P. chabaudi chabaudi AS (Pcc) using PbT-II cells, which recognize a common epitope of these parasites. In the acute phase of infection, CD4+ T-cell responses in PbA-infected mice showed a higher involvement of Th1 cells and a lower proportion of Ly6Clo effector CD4+ T cells than those in Pcc-infected mice. Transcriptome analysis of PbT-II cells indicated that type I interferon (IFN)-regulated genes were expressed at higher levels in both Th1- and Tfh-type PbT-II cells from PbA-infected mice than those from Pcc-infected mice. Moreover, IFN-α levels were considerably higher in PbA-infected mice than in Pcc-infected mice. Inhibition of type I IFN signaling increased PbT-II and Ly6Clo cell numbers and partially reversed the Th1 over Tfh bias of the PbT-II cells in both PbA- and Pcc-infected mice. However, in the memory phase, we did not find a significant difference in the phenotype of PbT-II cells between PbA- and Pcc-primed mice. These observations suggested that the differences in Plasmodium-specific CD4+ T-cell responses between PbA- and Pcc-infected mice were at least partially associated with the difference in type I cytokine production during the early phase of the infection.

overall_design: PbT-II cells underwent adoptive transfer into B6 mice and infected with Plasmodium berghei ANKA. Seven days after infection, we sorted subsets of PbT-II cells based on their expression of CD11a and CD49d expression.

last: Ntita

first: Shin-Ichi

last: Inoue

first: Jiun-Yu

last: Jian

first: Ganchimeg

last: Bayarsaikhan

first: Kazumi

last: Kimura

first: Daisuke

first: Mana

last: Miyakoda

first: Eriko

last: Nozaki

first: Takuya

last: Sakurai

first: Daniel

last: Fernandez-Ruiz

first: William

middle: R

last: Heath

first: Katsuyuki

last: Yui


title: Transcriptomic profile of trametinib on LPS-mediated gene expression in murine peritoneal macrophages

status: Public on Jul 12 2021

city: Wuhan

first: Jie

last: Wu

state: Hubei

address: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1277

department: Department of Cardiovascular Surgery

country: China

summary: To determine the underlying mechanisms by which trametinib affected LPS-induced inflammatory response in macrophages, we performed microarrays to define the global gene expression in murine peritoneal macrophages treating with trametinib or vehicle followed by LPS stimulation.

overall_design: We injected three mice with thioglycollate broth intraperitoneally. Three days later, the mice were injected with 5 ml of cold PBS into the peritoneal cavity and gently massaged. Then, the peritoneal fluid was collected, centrifuged, and the cell pellet was resuspended in DMEM supplemented with 10% FBS and cultured in 6-well-plate at 37℃. After adhering for 1h, the cells were gently washed with warm PBS for 3 times to remove non-adherent cells and then cultured with fresh medium. After additional 24h of culture, these cells were treated with trametinib (100nM) or vehicle (DMSO) for 2h, and then challenged by LPS (100ng/ml) for 12h.

first: Shanshan

last: Chen

first: Heng

last: Xu


title: Epigenetic analysis of perinatal maturation of supporting cells in the organ of Corti

first: Neil

last: Segil

summary: Sensory hair cells cannot be regenerated through transdifferentiation of neighboring supporting cells in the organ of Corti in mature mammalian animals, but limited regeneration capacity exists in supporting cells at neonatal stage in mouse and this transdifferentiation potential is rapidly lost during the first week of postnatal maturtion. We hypothesized that epigenetic decommissioning of hair cell gene enhancers in supporting cells during postnatal maturation leads the permanent silencing of hair cell genes and the loss of transdifferentiation potential. To test this hypothesis, we FACS purified hair cells and supporting cells from cochleae at different developmental stages for transcritomic analysis (RNAseq), chromatin accessibility assay (ATACseq) and histone modification profiling (ChIPseq or CUT&RUN). We first defined hair cell genes and predicted their potential active enhancers. We found that hair cell gene promoters and enhancers were kept in a primed-but-silenced status (H3K4me1/3+, low H3K27ac but high H3K27me3) in supporting cells at neonatal stage. During postnatal maturation, hair cell gene enhancers are decommissioned through H3K4me1 removal, leading to the permanent silencing of hair cells genes. We also found that hair cell gene enhancer decommissioning process correlated with the base-to-apex wave of transdifferentiation potential loss. In addition, hair cell gene enhancer commissioning status is preserved in mature utricular supporting cells, which can regenerate hair cells through transdifferentiation even at adult stage. Those data together suggest that decommissioning of hair cell gene enhancers in supporting cells during postnatal maturation is the epigenetic mechanism underlying the loss of regeneration capacity in the organ of Corti.

overall_design: Sensory hair cells (Atoh1-GFP+) were purified from postnatal day 1 (P1) animals for RNAseq, ATACseq, ChIPseq or CUT&RUN for hair cell gene identification and putative active enhancer prediction. Supporting cells (Lfng-GFP+) were purified from embryonic day 17.5 (E17.5), P1 and P6 animals for RNAseq, ATACseq, ChIPseq and CUT&RUN to interrogate the epigenetic modification changes surrounding hair cell gene regulatory elements during perinatal matruation. To compare the epigenetic status of supporting cells between apical half and basal half of cochlea, organs were cut into two halves after dissection and then supporting cells were purfied from each half for H3K4me1 CUT&RUN. To compare enhancer commissioning status in cochlear and utricular supporting cells at mature stage, supporting cells were purified from cochleae or utricles at 3 week old stage for H3K4me1 profiling by CUT&RUN and chromatin accessibility assay by ATACseq.

first: Litao

last: Tao


title: Local Delivery of mRNA Encoding Cytokines Promotes Antitumor Immunity and Tumor Eradication Across Multiple Preclinical Tumor Models

status: Public on Jul 10 2021

first: Joon Sang

last: Lee

state: MA

address: 270 Albany Street

department: Oncology

institute: Sanofi

summary: Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or anti-vector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin (IL)-12 single chain, interferon (IFN)-α, granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors as well as disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

overall_design: Subcutaneous B16F10 tumors in C57BL6 mice (n=5/group) were treated with a single intratumoral injection of cytokine mRNA mixture (80 µg total, 20 µg per mRNA component) encoding a murine single chain IL-12, murine IL-15sushi, murine GM-CSF and murine IFNalpha or control mRNA (80 µg luciferase mRNA) .  Tumors were collected  7 days following intratumoral mRNA injection and subjected to global gene expression to assess changes between cytokine mRNA and control treated tumors.

first: Timothy

last: Wagenaar

first: Natalia

last: Malkova

first: Hui

last: Cao

first: Jack

last: Pollard

first: Joon

middle: S


title: Small intestinal tissue and epithelial regeneration after 12 Gy whole-body irradiation of C57BL/6 wild type mice

city: Amsterdam

first: Sander

last: Meisner

state: NH

address: Meibergdreef 69-71

department: Tytgat institute

country: Netherlands

institute: AMC

summary: C57BL/6 wild type mice were exposed to 12 Gy whole-body irradiation. Before irradiation (t=0) and at t=24, 48 and 96 hours after irradiation, mice (n=10 per time point) were euthanized. From n=5 mice per time point RNA was isolated from small-intestinal (jejunum) tissue, from n=5 mice per time point RNA was isolated from EDTA-isolated epithelial cells from small-intestinal tissue (jejunum).

overall_design: n=40 in total: n=5 small intestinal tissue per time point, n=5 EDTA-isolated small intestinal epithelial cells per time point. Time points t=0h, 24h, 48h, 96h

first: Jonathan

middle: H

last: van der Meer

first: Vanesa

last: Muncan


title: An isoform of Dicer protects mammalian stem cells against multiple RNA viruses

city: London

first: Enzo

last: Poirer

address: 1 Midland Road

country: United Kingdom

institute: Francis Crick Institute

summary:
In mammals, early resistance to viruses relies on interferons, which protect differentiated but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialised Dicer protein that cleaves viral double stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. Here we identify an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses, including Zika virus and SARS-CoV-2, by dicing viral double-stranded RNA to orchestrate antiviral RNAi.
Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity in which different cell-intrinsic antiviral pathways are tailored to the differentiation status of cells.

overall_design: Mouse brain organoids were infected with ZIKV or SARS-CoV-2 for 4 days. RNA was extracted from infected organoids and deep-sequenced.

middle: Z

last: Poirier

first: Caetano

last: Reis e Sousa


title: Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer

city: New York

first: Gregory

last: Putzel

state: NY

address: 430 East 29th Street

department: Microbiology

institute: NYU Langone Health

summary: Group 3 innate lymphoid cells (ILC3s) are critical regulators of intestinal homeostasis, yet their role in cancer remains elusive. Here, we identify that the tumor microenvironment of humans and mice with colorectal cancer (CRC) contains altered ILC3 responses that resemble those found during intestinal inflammation and are characterized by reduced frequencies, altered subset distribution, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that ILC3-specific major histocompatibility complex class II (MHCII) was required to limit the progression of CRC. Interestingly, ILC3-specific MHCII also prevented resistance to anti-PD-1 immunotherapy by regulating intestinal inflammation and microbiota composition. Finally, humans with intestinal inflammation and dysregulated ILC3s also harbor specific microbiota that are sufficient to promote immunotherapy resistance in mice. Collectively, these data define a protective role for antigen-presenting ILC3s in cancer, and indicate that inherent disruption of this pathway drives CRC progression and immunotherapy resistance.

overall_design: Human ILC3s (CD45+CD3-CD4-CD8-CD19-CD94-CD14-CD123-FcεRIa-CD34-CD11c-CRTH-2-CD127+CD117+) were sort-purified from surgical-resection samples from the colon or ileum of patients with colon cancer or IBD. Sorted cells were used to prepare RNA sequencing libraries by the Epigenomics Core at Weill Cornell Medicine, using the Clontech SMARTer Ultra Low Input RNA Kit V4 (Clontech Laboratories). Sequencing was performed on an Illumina HiSeq 4000, yielding 50-bp single-end reads. Raw sequencing reads were demultiplexed with Illumina CASAVA (v.1.8.2). Adapters were trimmed from reads using FLEXBAR (v.2.4) and reads were aligned to the NCBI GRCh37/hg19 human genome using the STAR aligner (v.2.3.0) with default settings. Reads per gene were counted using Rsubread (Liao et al., 2019). Prior to differential expression analysis, genes were prefiltered, keeping only those genes with 50 or more counts in at least two samples. Differential expression analysis was performed using DESeq2 version 1.20.0 (Love et al., 2014) using both site (tumor/adjacent) and patient ID as factors in the design. A false discovery rate of 0.1 was taken to indicate significance. Principal components analysis (PCA) was performed using the top 500 highest variance genes after applying DESeq2’s variance stabilizing transformation. The degree to which samples clustered by site (tumor versus adjacent) in the PCA was assessed using PERMANOVA (Anderson, 2001) as implemented by the adonis function of the vegan R package (Oksanen et al., 2019) using the Euclidean metric and 20,000 permutations.

first: Jeremy

last: Goc

last: Sonnenberg


title: Analysis of 2-cell embryos resulted from COPS3 protein inactivation in pronuclear-stage mouse oocytes

city: Muenster

first: Michele

last: Boiani

address: Roentgenstrasse 20

country: Germany

institute: Max Planck Institute Muenster

summary:
In metazoans, maternal factors deposited in the ooplasm during oocyte growth are largely responsible for the control of initial development of the newly formed embryo. The gradual handover from oocytic to embryonic control, known as oocyte-to-embryo or maternal-to-zygotic transition, involves de novo synthesis of new embryonic factors (embryonic genome activation) as well as the timely degradation of preexisting maternal factors deposited in oocytes as transcripts or proteins. Nevertheless, the role of the protein deposits largely remains undeciphered. In order to study these deposits, genetic knockouts and knockdown systems are not suited because they do not react with the protein that has already been deposited. Targeted proteolysis can be accomplished with the 'Trim-away' method (PMID 29153837). Building on our proteomic datasets and our own adaptation of the Trim-away method (PMID 31638890), we demonstrate a large deposit of COP9 signalosome complex subunit 3 (COPS3) protein in mouse oocytes, and its critical role during the oocyte-to-embryo transition. COPS3 protein depletion in zygotes causes 2-cell arrest, at variance with the original report describing early postimplantation lethality (PMID 12972600). We conclude that peri-implantation demise was possibly the second time when the gene function was needed in development, while earlier functions had been hidden by maternal deposits.
In the Trim-away method (PMID 29153837) a cell expressing the ubiquitin-protein ligase TRIM21 is supplied e.g. injected with a specific antibody to a protein of interest. As a result, the ternary complex (target protein-antibody-TRIM21) is destroyed in the proteasome. To test for the requirement of COPS3 in early mouse development, we chose to deplete COPS3 using its antibody in conjunction with TRIM21. To distinguish between depletion and inhibition of the target protein, we also tested the antibody alone. In total, we examined four groups, as follows. Pronuclear (PN)-stage oocytes (B6C3F1 x CD1) were microinjected with 100 picoliters of mix comprised of: Trim21 mRNA 0.2 mg/mL + dextran beads as tracer (named 'group 2');  Trim21 mRNA 0.2 mg/mL + anti-COPS3 antibody 0.5 mg/mL + dextran beads as tracer (named 'group 3'); anti-COPS3 antibody 0.5 mg/mL + dextran beads as tracer (named 'group 4'). Antibody was ab79698 from AbCam, column-purified twice to remove preservatives. These groups were compared with non-injected PN oocytes (named 'group 1'). Each group was produced in duplicate (replicate '.0' and replicate '.1'). On the day after microinjection, late 2-cell embryos were collected and lysed for transcriptome analysis. Zygotes of groups 3 and 4 presented stunted progression to the 2-cell stage, in contrast to the almost full rates of the zygotes of groups 1 and 2. Transcriptome analysis revealed that embryos group 2 are very similar to those of group 1 (97 differently expressed mRNAs), whereas embryos of groups 3 and 4 differ from those of group 1 by 3499 and 973 mRNAs, respectively (fold change ≥2, p≤0.05; Student’s t test).

overall_design: Groups 1,2,3,4 of zygotes were fertilized in vivo and treated on different days of the same week, and cultured for 28 hours, however the two replicates of each group were from the same day. RNA was extracted and analyzed at the late 2-cell stage.

first: Steffen

last: Israel


title: Proteomic analysis of the pathogen surface in cholera identifies host proteins involved in defense and microbiota recognition

status: Public on Jul 08 2021

city: Boston

first: Matthew

last: Waldor

address: 181 Longwood Avenue

institute: Harvard Medical School

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Mus musculus
Vibrio cholerae H1

platform_organism:
Mus musculus
Vibrio cholerae H1

overall_design: Refer to individual Series


title: ATACseq, CutnRun and RNAseq data from Merkel cell and Touch dome epithelial progenitor cells, from wild type and Pou4f3-mutant epidermis

first: Neil

last: Segil

summary: The role of POU4F3 in Merkel cell maturation, but not induction, is similar to its role in hair cells. This prompted us to investigate whether POU4F3 also regulates the accessibility of ATOH1 targets in a feed-forward manner during the maturation of Merkel cells.  Our results suggest that, just as in hair cells, the pioneer factor activity of POU4F3 is also required for ATOH1 to correctly coordinate mechanosensory differentiation in Merkel cells.

overall_design: Epithelial progenitors surrounding Merkel cells in touch domes in E17.5 epidermis can be isolated by their combinatory expression pattern of Integrin-α6, Cd34, Sca-1, and CD200 (Doucet et al., 2013), whereas Merkel cells can be isolated by their expression of ATOH1-GFP. We used ATOH1-GFP mice and ATOH1-GFP; Pou4f3 mutant mice to purify touch dome epithelial progenitors and Merkel cells from wild type and Pou4f3-mutant epidermis. We analyzed accessible chromatin with μATACseq and identified elements binding ATOH1 with CUT&RUN. We identified 4,683 ATOH1 target elements that were accessible in nascent Merkel cells, but not epithelial progenitors. We found that the accessibility of 710 of these 4,683 de novo elements was dependent on POU4F3. Many of these POU4F3-dependent de novo ATOH1-target elements were near genes critical for Merkel cell maturation and mechanotransduction, such as Krt20, Rab3c (Haeberle et al., 2004; Perdigoto et al., 2014), and the light-touch mechanotransduction channel Piezo2 (Ikeda et al., 2014; Maksimovic et al., 2014; Ranade et al., 2014; Woo et al., 2014). RNAseq confirmed that expression of Piezo2, Rab3c and Krt20 was significantly reduced in Pou4f3 mutant Merkel cells (FDR < 0.1).

first: Yu

last: Haoze

first: Tao

last: Litao

first: Segil

last: Neil


title: Proteomic analysis of the pathogen surface in cholera identifies host proteins involved in defense and microbiota recognition [II]

summary: The microbial cell surface is a critical site of microbe-host interactions that often control infection outcomes. Defining the set of host proteins present at this interface has been challenging. Here, we used a surface biotinylation approach coupled to quantitative mass spectrometry to identify and quantify both bacterial and host proteins present on the surface of diarrheal fluid-derived V. cholerae in an infant rabbit model of cholera. Our data showed that V. cholerae surfaces were coated with numerous host proteins, whose abundance were driven by the presence of cholera toxin, including the C type lectin SP-D. Mice lacking SP-D had enhanced V. cholerae intestinal colonization. Additional host proteins (AnxA1, LPO and ZAG) capable of binding V. cholerae were also found to recognize distinct taxa of the murine intestinal microbiota, suggesting that these host factors may play roles in intestinal homeostasis in addition to host defense. Proteomic analysis of microbial surfaces is valuable for identifying host interactions that regulate infection and homeostasis with both pathogens and endogenous microbiota alike.

overall_design: We performed RNAseq on V. cholerae in vitro in presence of SP-D or denatured SP-D.

first: Abdelrahim

last: Zoued

first: Hailong

last: Zhang

first: Ting

first: Rachel

middle: T

last: Giorgio

first: Carole

middle: J

last: Kuehl

first: Bolutife

last: Fakoya

first: Brandon

last: Sit


title: µATACseq: ATACseq with as few as 100 cells

summary: μATACseq data generated from 100, 500 and 1,000 FACS-purified Atoh1-GFP expressing cerebellar granule precursors (CGPs) are consistent with those from 20,000 CGPs by the standard ATACseq protocol (R > 0.85), with comparable percentages of both the reads mapped to the promoters (±1000bp Transcription Start Sites), and to the distal elements. Of the peaks identified from the standard ATACseq method, more than 70% are detected among those from the μATACseq with as low as 100 cells. In addition, μATACseq lowers the sequencing cost by generating 10-fold fewer mitochondrial reads and up to 1.7-fold fewer PCR duplicates. Surprisingly, the μATACseq protocol is robust over a 10-fold difference in the transposase-to-cell ratios measured by (1) the fragment size distribution inferred from pair-end sequencing, (2) the depth of the predicted transcription factor footprints, (3) and the concordance between biological replicates. Further more, μATACseq is fast and convenient, does not require multiple washing steps or nuclear isolation, and demands only 20 minutes prior to DNA purification and sequencing library amplification.

overall_design: µATACseq was validated using CGPs since they are aboudant and they express ATOH1-GFP. FACS-purified cells (100, 500,1000 cells per reaction) were centrifuged to remove the supernatant. 20 μL lysis buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF, 0.2% N-P40) was added, followed by pipetting 6-10 times to release the nuclei. 30 μL reaction buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF, 1000 unit transposome) was added directly to the cell lysis, and mixed by vortexing. The reaction was incubated at 37 °C for 20 minutes. 350 μL ERC buffer (MiniElute, Qiagen) was added to stop the reaction. After DNA purification (MiniElute, Qiagen), ATACseq libraries were constructed as previously described (Buenrostro et al., 2013).


title: Transcriptomic and epigenetic analysis of neonatal supporting cells during Notch inhibition-induced transdifferentiation

summary: Latent regeneration potential has been found in neonatal supporting cells in the organ of Corti in mouse. Upon Notch inhibition by DAPT, postnatal day 1 (P1) supporting cells transdifferentiate into hair cells. To profile the transcriptomic and epigenetic changes in supporting cells during transdifferentiation, we FACS purified supporting cells from DAPT treated cochleae for RNAseq, ATACseq and H3K27ac CUT&RUN. We found that hair cell genes were up-regulated in supporting cells after DAPT treatment and that hair cell gene induction was accompanied by epigentic activation of hair cell gene cis-regulatory elements through chromatin accessibility increase and H3K27ac accumulation, suggesting the regulatory roles of epigenetic activation of hair cell gene elements for hair cell gene expression induction.

overall_design: P1 cochlear organs were cultured and treated by DAPT or DMSO. At 24 hour post treatment, all supporting cells (Lfng-GFP+) were purified for RNAseq. At 48 hours post treatment, supporting cells undergoing transdifferentiation upon DAPT (permissive, Lfng-CreER/TdT+  and Atoh1-GFP+), supporting cells irresponsive to DAPT (nonpermissive, Lfng-CreER/TdT+ only) and DMSO-treated supporting cells (control, Lfng-CreER/TdT+ only) from control organs were purified for transcriptomic analysis (RNAseq), chromatin accessibility assay (ATACseq) and H3K27ac profile (CUT&RUN).

first: Litao

last: Tao


title: Human brain organoids assemble functionally integrated bilateral optic vesicles

city: Goettingen

first: Argyris

last: Papantonis

address: Robert-Koch-Str 40

department: Department of Pathology

institute: University Medical Center Goettingen

summary: During embryogenesis, optic vesicles develop from the diencephalon via a complex process of organogenesis. Using iPSC-derived human brain organoids, we attempted to simplify the complexities and demonstrate the formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day thirty, brain organoids could assemble optic vesicles, which progressively develop as visible structures within sixty days. These optic vesicle-containing brain organoids (OVB-Organoids) constitute a developing optic vesicle’s cellular components, including the primitive cornea and lens-like cells, developing photoreceptors, retinal pigment epithelia, axon-like projections, and electrically active neuronal networks. Besides, OVB-Organoids also display synapsin-1, CTIP-positive, myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photoreceptor activity of OVB-Organoids, and light sensitivities could be reset after a transient photo bleach blinding. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow conducting interorgan interaction studies within a single organoid.

overall_design: IMR90 derived normal neural and OVB organoids (3 biological replicates per condition) were collected for RNA extraction and sequencing analysis.

first: Natasa

last: Josipovic


title: Genetic screen for suppressors of lncRNA-mediated transcription interference identifies a gain-of-function mutation in the essential Pol2 termination factor Seb1

first: Angad

last: Garg

state: New York

department: Molecular Biology

institute: Memorial Sloan Kettering Cancer Center

summary: This RNA-Seq analysis compares gene expression of the STF-19 (seb1-G476S gain-of-function mutant) compared to a seb1+ WT in the fission yeast S. pombe. Both strains had WT-rpb1 with a TPI1 terminator sequence and a 3' selection marker.

overall_design: 1 test sample was analyzed. 1 control sample sequenced within the same run was previously deposited under GEO accession no. GSE160852. Each sample has three biological replicates.

first: Beate

last: Schwer

first: Stewart

last: Shuman


title: Transcriptomic analysis of neonatal supporting cells after histone modification perturbations

first: Neil

summary: At neonatal stage, sensory hair cell gene promoters and enhancers are primed by H3K4me3/me1 in neighboring supporting cells, but silenced by deacetylation and H3K27 tri-methylation. To test whether this primed-but-silenced epigenetic status leads to the repressed expression of hair cell genes in supporting cells, we FACS-purified Lfng-GFP+ supporting cells from cultured postnatal day 1 (P1) ochlear organs 48 hours after TSA (HDAC inhibitor) or GSK-343 (EZH2 inhibitor) for transcritomic analysis by RNAseq. We found that hair cell genes could be induced by either blocking of histone deacetylation or inhibition of H3K27 trimethylation, suggesting histone deacetylation and H3K27 trimethylation are required to repress hair cell gene expression in supporting cells at neonatal stage when a latent transdifferentiation potential still exists in supporting cells.

overall_design: Transcriptomes of supporting cells purified from DMSO, TSA or GSK-343 treated organs were analyzed by RNAseq


title: 48hr 2a3 vs YTS177 CD4+ PLN T cell Microarray

city: Chapel Hill

first: Matthew

last: Clark

state: NC

address: 125 Mason Farm Road

department: Microbiology and Immunology

institute: University of North Carolina at Chapel Hill

summary: BDC2.5 mice treated with 1mg isotype control (2a3) or anti-CD4 (YTS177) antbodies for 48hrs. PLN CD4+ T cells sorter for microarray analysis.

overall_design: 48hr in vivo treatment with 1mg isotype control (2a3) or anti-CD4 (YTS177) antbodies

first: Aaron

last: Martin

first: Roland

last: Tisch


title: Cilium induction triggers differentiation of glioma stem cells

summary: Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of the fundamental biology of GSCs is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit an elevated level of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components at the ciliary base is sufficient to induce ciliogenesis in a subset of GSCs via sequestering PDGFR-α from its original location to newly induced cilium. Importantly, restoring ciliogenesis caused GSCs to behave like healthy NPCs switching from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a crucial role for cilium as a molecular switch in determining GSCs' fate and suggest that cilium induction is an attractive strategy to intervene in GSCs proliferation.

overall_design: Control and Nek2 knock-down glioblastoma patient-derived GSC line U3047MG (PDGFRA, OPC-like) was subjected to RNA-sequencing in two biological replicates.


title: Using Mouse Embryonic Fibroblast to test the pioneer factor activity of POU4F3

last: Segil

summary: Previous unbiased screening for the nucleosome binding affinity of transcription factors predicts that POU domain transcription factors have strong nucleosome binding potential. Here using reprogramming strategy we found that POU4F3 has pioneer factor activity at ATOH1 target elements that require POU4F3 for their accessibility.

overall_design: We overexpressed ATOH1, POU4F3, ATOH1+POU4F3 and a GFP control in mouse embryonic fibroblasts (MEFs), and used CUT&RUN and μATACseq to assess the transcription factor binding and the resulting chromatin structure at the 1,458 ATOH1 targets that required POU4F3 for their accessibility in hair cells. We found that POU4F3 alone was able to bind most of these distal elements within closed chromatin in MEFs, demonstrating pioneer factor activity. The presence of POU4F3 also enabled ATOH1 to recognize and bind these targets in MEFs. Surprisingly, although overexpression of ATOH1+POU4F3 resulted in the opening of a small number of these distal elements in MEFs (233 / 1,458), the majority remained closed (963 / 1,458), but were nevertheless bound by both transcription factors, suggesting that additional hair cell-specific transcription factors / co-factors, which are not present in MEFs, are required for the selective opening and activation of this group of distal elements.

first: Yu

last: Haoze

first: Tao

last: Litao

first: Segil


title: Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology

city: Kyoto

first: Shimpei

last: Gotoh

address: 53 Kawaharacho, Shogoin, Sakyo-ku

department: Center for iPS cell Research and Application

institute: Kyoto University

summary: Fluid shear stress in the airway chip culture promotes induction of iPS cell-derived multiciliated airway cells (iMCACs) and helps establish the global axis of planar cell polarity.

overall_design: Human iPS cell-derived airway epithelial progenitor cells in 3D Matrigel blocks were dissociated on Day 28 and placed on the laminin-coated membrane of the 12-well cell culture inserts or of the upper channel of the devices. In "Healthy donor-iMCACs fluid shear stress(+)", the attached cells 12 hours after seeding were perfused with culture medium using a micro peristaltic pump until Day 42. In "Healthy donor-iMCACs fluid shear stress(-)" and "Healthy donor-iMCACs cell culture insert", the cells were not perfused with culture medium. All the cells in each condition were harvested on Day 42. The transcriptomes of these cells were sequenced with paired-end reads (150-bp) on an Illumina Novaseq 6000 instrument.

first: Naoyuki

last: Sone


title: Mapping the Cellular Origin and Early Evolution of Down Syndrome Leukemogenesis [miRNA-Seq]

city: Toronto

first: Elvin

last: Wagenblast

state: ON

address: 101 College Street

department: Princess Margaret Cancer Centre

country: Canada

institute: UHN

type: Non-coding RNA profiling by high throughput sequencing

summary: Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia. As Down syndrome leukemogenesis initiates during fetal development, we sought to characterize the cellular mechanisms of preleukemic initiation and leukemic progression using CRISPR/Cas9-mediated gene editing in human disomic and trisomic fetal liver hematopoietic cells and xenotransplantation. Compared to disomic fetal liver, trisomy 21 initiated atypical fetal hematopoiesis, in part through up-regulation of chromosome 21 miRNAs. GATA1 mutations caused transient preleukemia only when introduced into trisomy 21 long-term hematopoietic stem cells. By contrast, progression to leukemia was independent of trisomy 21 and originated in a wide spectrum of stem and progenitor cells through additional mutations in cohesin genes such as STAG2. CD117+ cells mediated the propagation and progression of the preleukemic and leukemic disease. Treatment with small molecule CD117/KIT inhibitors efficiently targeted preleukemic stem cells and blocked progression to leukemia; thereby laying the groundwork for early prevention strategies in Down syndrome newborns.

overall_design:
We profiled miRNAs from N-FL and T21-FL CD34+ enriched HSPCs by miRNA-Seq.
*** Submitter declares raw data will be uploaded to EGA, due to privacy concerns.

first: John

middle: E

last: Dick

first: Eric

last: Lechman

first: Alex

last: Murison

first: Sajid

middle: A

last: Marhon

first: Martino

last: Gabra


title: Mapping the Cellular Origin and Early Evolution of Down Syndrome Leukemogenesis [CUT&RUN]

overall_design:
We performed Cut&Run assays to profile genome-wide GATA1 binding sites and also to quantify binding changes upon GATA1s editing in N-FL and T21-FL CD34+ enriched HSPCs.
*** Submitter declares raw data will be uploaded to EGA, due to privacy concerns.


title: Proteomic analysis of the pathogen surface in cholera identifies host proteins involved in defense and microbiota recognition [I]

last: Waldor

overall_design: We performed RNAseq on proximal and distal small intestine tissues derived from 4 V. cholerae-infected WT and 4 V. cholerae-infected Sftpd KO mice to identify differentially expressed genes during infection.

first: Abdelrahim

last: Zoued

first: Hailong

last: Zhang

first: Ting

first: Rachel

last: Giorgio

first: Carole

last: Kuehl

first: Bolutife

last: Fakoya

first: Brandon

last: Sit


title: A SUMOylation-induced Satb1/Satb2 switch drives embryonic stem cell differentiation [HiC]

city: Freiburg

first: Pierre

middle: Daniel

last: Cauchy

state: Baden-Württemberg

address: Breisacher Str. 115

department: Zentrum für Translationale Zellforschung

institute: University Medical Center Freiburg

summary: The chromatin organizers Satb1 and Satb2 regulate developmental genes in a tissue- and locus-specific manner. In mouse Embryonic Stem Cells (mESCs), the Satb proteins are involved in the balance between pluripotency and differentiation by direct control of key developmental factors such as Nanog and a number of Hox genes. Despite their structural similarities, the Satb proteins regulate mESC pluripotency in opposing ways: Satb1 promotes differentiation by repressing Nanog and activating the neural genes Bcl2 and Nestin, while Satb2 supports the pluripotent state by activating Nanog and repressing Satb1. To further address the mechanisms by which the Satb proteins regulate gene expression, we examined the conjugation of Satb2 with the small ubiquitin-like modifier (SUMO) in pluripotent and differentiated mESCs. We describe for the first time the endogenous SUMOylation of Satb2 in mESCs as a response to developmental cues. We found that Satb2 is progressively SUMO2-modified during differentiation of ESCs towards ectodermal precursors. Moreover, we identified Zfp451 as a SUMO E3 ligase able to interact with and modify Satb2 with SUMO2 in vitro and in vivo. Ablation of Zfp451 or mutation of the SUMO-acceptor lysines in the Satb2 protein disrupt the ability of mESCs to efficiently shut-down pluripotency genes and activate the differentiation program. Importantly, forced expression of SUMO2-Satb2 N-terminal fusions rescues the differentiation defect of SUMO-Satb2 deficient mESCs. Mechanistically, SUMOylation reduces binding of Satb2 to a subset of loci associated to pluripotency genes and changes the composition of Satb2-containing complexes in chromatin. Taken together, our data suggests that SUMO modification of the chromatin organizer Satb2 by the E3 ligase Zfp451 is required for the efficient downregulation of pluripotency genes and initiation of the differentiation program in mouse embryonic stem cells.

overall_design: HiC during mESC differentiation in neural progenitor conditions, with and without Satb2 K233/350R sumoylation mutant

first: Gustavo

last: Urrutia

first: Haribaskar

last: Ramachandran

first: Senthilkumar

last: Ramamoorthy

first: Clapes

last: Thomas

first: Boller

last: Sören

first: Gerhard

last: Mittler

first: Rudolf

last: Grosschedl


title: Inducible and reversible inhibition of miRNA-medaited gene repression in vivo

status: Public on Jul 07 2021

first: Andrea

last: Ventura

department: Cancer Biology and Genetics

summary: Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

overall_design: RNAseq analysis of gene expression in a panel of tissues from control and t6b expressing mice

first: Xiaoy

last: Li

first: Gasapre

last: La Rocca

first: Bryan

last: King


title: Proneural genes define ground state rules to regulate neurogenic patterning and cortical folding

city: Bethesda

first: Matthew

last: Brooks

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

institute: NIH

summary: Asymmetric neuronal expansion is thought to drive evolutionary transitions from lissencephalic to gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes interact to sustain neurogenic continuity and lissencephaly in rodents. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage-restricted due to Neurog2-Ascl1 cross-repression, and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selective killing of double+ NPCs using Neurog2-Ascl1 split-Cre mice and three ‘deletor’ strains breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Consistent with proneural genes driving discontinuous neurogenesis and folding via Notch, NEUROG2, ASCL1 and HES1 transcripts are modular in gyrencephalic macaque cortices. Neurog2/Ascl1 double+ NPCs are thus Notch-ligand expressing ‘niche’ cells that control neurogenic periodicity and cortical gyrification.

overall_design: CD15+ GFP/mCherry negative, single, double positive cortical progenitors were isolated from mouse E12.5 Neurog2Flag-mcherryKI/+;Ascl1GFPKI/+ dorsal telencephalic tissue by Fluorescence-activated cell sorting (FACS). Total RNA from each cell types was extracted by Trizol LS (Life Technologies). RNAseq was performed on each cell type after constructing mRNA libraries using the Illumina TruSeq Stranded mRNA kit. Each library was paired-end sequenced to a length of 2x125 bases. Fastq files were generated from reads passing chastity filter.

first: Sisu

last: Han

first: Anand

last: Swaroop

first: Carol

last: Schuurmans


title: Transcriptome Stim1 R304W skeletal muscle

city: ILLKIRCH

first: Jocelyn

last: Laporte

address: 1, rue Laurent Fries

country: France

institute: IGBMC

summary: Comparative RNAseq of tibilias anterior from Stim1 R304W and WT male mice at 4 months

overall_design: The study is composed of 2 groups of mice: WT, Stim1R304W/+

first: Roberto

last: Silva-Rojas

first: Anne-Laure

last: Charles

first: Sarah

last: Djeddi

first: Bernard

last: Geny

first: Johann

last: Böhm


title: Murine ex vivo cultured alveolar macrophages provide a novel tool to study tissue-resident macrophage behavior and function

city: Vienna

first: Sylvia

last: Knapp

address: Waehringer Guertel 18-20

country: Austria

institute: Medical University of Vienna

summary: MexAMs are functionally similar to primary AMs, so we compared their  transcriptional profile to different macrophage populations. These included BMDMs, as the macrophage type predominantly used for in vitro studies, arising from myeloid bone marrow progenitor cells, as well as peritoneal macrophages (PMs), a mature TRM population exposed to a different tissue microenvironment.

overall_design: mRNA profiles of listed cell types (BALF, PLF, BMDM, mexAM)

first: Gorki

last: Anna-Dorothea


title: Identification and validation of circulating miRNAs as potential new biomarkers for severe liver disease in patients with leptospirosis

city: Bangkok

first: Natthaya

last: Chuaypen

state: Select a State or Province

address: 1873 Rama 4 road, Pathumwan

department: Biochemistry

country: Thailand

institute: Chulalongkorn University, Faculty of Medicine

summary:
Background: Leptospirosis, a global zoonotic infectious disease, has various clinical manifestations ranging from mild self-limiting illness to life-threatening with multi-organ damage, including liver involvement. This study was aimed at identifying circulating microRNAs (miRNAs) as novel biomarkers for predicting severe liver involvement in patients with leptospirosis.
Methods: In a discovery set, 12 serum samples of patients with anicteric and icteric leptospirosis at initial clinical presentation were used for miRNA profiling by a NanoString nCounter miRNA assay. In a validated cohort, top candidate miRNAs were selected and further tested by qRT-PCR in serum samples of 81 and 16 individuals with anicteric and icteric leptospirosis, respectively.
Results: The discovery set identified 38 significantly differential expression miRNAs between the two groups. Among these, miR-601 and miR-630 were selected as the top two candidates significantly up-regulated expressed in the icteric group. The enriched KEGG pathway showed that these miRNAs were mainly involved in immune responses and inflammation. In the validated cohort, miR-601 and miR-630 levels were significantly higher in the icteric group compared with the anicteric group. Additionally, these two miRNAs displayed good predictors of subsequent acute liver failure with a high sensitivity of 100%. On regression analysis, elevated miR-601 and miR-630 expression were also predictive of multi-organ failures and poor overall survival.
Conclusion: Our data indicated that miRNA expression profiles were significantly differentiated between the icteric and anicteric groups. Serum miR-601 and miR-630 at presentation could potentially serve as promising biomarkers for predicting subsequent acute liver failure and overall survival in patients with leptospirosis.

overall_design: A total of 12 serum samples were randomly selected from the stored samples to investigate the expression profile of 800 human miRNAs using the nCounter® Human v3 miRNA miRNA Expression Assays (NanoString Technologies, Seattle, USA).

first: Umaporn

last: Limothai

first: Pattapon

last: Kunadirek

first: Pornchai

last: Kaewsapsak

first: Patipark

last: Kueanjinda

first: Nattachai

last: Srisawat

first: Pisit

last: Tangkijvanich


title: RNAseq analysis of human CD4+ regulatory Tregs and Tconvs in COVID-19 and healthy donors isolated from peripheral blood.

first: CBDM

last: Lab

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

overall_design: Human Tregs and Tconvs from COVID-19 patients and healthy donors were magnetically isolated from frozen PBMCs using a Stem Cell kit, as CD4+CD25hiCD127lo and CD4+CD25–, respectively. Around 1,000 isolated cells were directly into lysis buffer for RNA-seq (Smart-seq2)  library preparation following the standard ImmGen low-input protocol (www.immgen.org). Samples were collected and sequenced in two cohorts.

first: Silvia

last: Galvan Pena

first: Juliette

last: Leon

first: Zachary

last: Manickas-Hill

first: Jonathan

last: Li

first: Diane

last: Mathis

first: Christophe

last: Benoist


title: A SUMOylation-induced Satb1/Satb2 switch drives embryonic stem cell differentiation [ChIP-Seq]

first: Pierre

last: Cauchy

overall_design: ChIP-Seq for Satb2, Satb1, and LSD1 during mESC differentiation in neural progenitor conditions, with and without Satb2 K233/350R sumoylation mutant

first: Gustavo

last: Antonio Urrutia

first: Haribaskar

last: Ramachandran

first: Senthilkumar

last: Ramamoorthy

first: Esen

last: Dogan

first: Gerhard

last: Mittler

first: Andrea

last: Pichler

first: Rudolf

last: Grosschedl


title: Anti-proliferative, Apoptosis and Anti-migration Properties on Androgen-independent Human Prostate Cancer by a Diarylpentanoid, MS13.

city: Selangor

first: Nurul Azwa

last: Abd. Wahab

state: Not US or Canada

address: Jalan Lagoon Selatan, Bandar Sunway, 47500 Subang Jaya, Selangor

department: Jeffrey Cheah School of Medicine and Health Sciences

country: Malaysia

institute: Monash university Malaysia

summary: Diarylpentanoids exhibit a high degree of anti-cancer activity and stability in vitro over curcumin in prostate cancer cells. Hence, this study aims to investigate the effects of a diarylpentanoid, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13) on cytotoxicity, anti-proliferative, apoptosis-inducing, anti-migration properties, and the underlying molecular mechanisms on treated androgen-independent prostate cancer cells, DU 145 and PC-3. A cell viability assay has shown greater cytotoxicity effects of MS13-treated DU 145 cells (EC50 7.57 ± 0.2 µM) and PC-3 cells (EC50 7.80 ± 0.7 µM) compared to curcumin (EC50: DU 145; 34.25 ± 2.7 µM and PC-3; 27.77± 6.4 µM). In addition, MS13 exhibited significant anti-proliferative activity against AIPC cells compared to curcumin in a dose- and time-dependent manner. Morphological observation, increased caspase-3 activity, and reduced Bcl-2 protein levels in these cells indicated that MS13 induces apoptosis in a time- and dose-dependent.  Moreover, MS13 effectively inhibited the migration of DU 145 and PC-3 cells. Our results suggest that cell cycle-apoptosis and PI3K pathways were the topmost significant pathways impacted by MS13 activity. Our findings suggest that MS13 may demonstrate the anti-cancer activity by modulating DEGs associated with the cell cycle-apoptosis and PI3K pathways, thus inhibiting cell proliferation and cell migration and inducing apoptosis in AIPC cells.

overall_design: The experiments were performed with three controls and three treated samples for each cell lines, DU 145 and PC-3. Three biological replicates were analyzed and three technical repeats were performed per sample.

first: Nurul

last: Wahab

first: Faridah

last: Abas

first: Iekhsan

last: Othman

first: Rakesh

last: Naidu


title: A SUMOylation-induced Satb1/Satb2 switch drives embryonic stem cell differentiation

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: A SUMOylation-induced Satb1/Satb2 switch drives embryonic stem cell differentiation [RNA-Seq]

overall_design: RNA-Seq in Satb2WT, Satb2K233/350R sumoylation double mutant, ZNF451WT, ZNF451KO cells during mESC differentiation in neural progenitor conditions


title: Genome-wide analysis of RNA polymerase II occupancy during HSV-1 infection in the presence and absence of ICP4.

city: Boulder

first: Jennifer

last: Kugel

state: Colorado

address: 3415 Colorado Ave

institute: University of Colorado at Boulder

summary: The goal of this study was to identify how the occupancy of RNA polymerase II (Pol II) on the host genome changes during HSV-1 infection and is impacted by the viral immediate early protein ICP4.  Pol II ChIP-seq experiments after infection with the wild-type (WT) virus and mutant ICP4 (n12) virus, compared to mock infection, revealed global increases and decreases in Pol II occupancy on the host genome that depended upon ICP4.

overall_design: RNA polymerase II ChIP-seq in HEK 293 cells after infection with wild-type HSV-1 virus, n12 virus (lacking functional ICP4), and mock infection as the control sample. ChIP-seq under all conditions was performed in biological replicate. Input chromatin from mock and infected cells was also sequenced.

first: Thomas

last: Rivas

first: James

last: Goodrich

middle: F


title: Gene expression profiles of  WDR4 knockdown in HCC cells

city: wuhan

first: xia

last: peng

state: hubei

address: zhongnan hospital

institute: wuhan university

summary: HCC cell line, Huh-7 cells and HCC-LM3 cells, was transfected with sh-WDR4-2 or sh-NC for 48hr to knockdown WDR4 expression, and check the downstream mRNA changes.

overall_design: Comparison of transcriptomes from sh-WDR4/sh-NC  in HCC cells

first: Peng

last: Xia

first: Hao

last: Zhang


title: Expression data of antibody fragments from Saccharomyces cerevisiae

status: Public on Jul 06 2021

city: Gothenburg

first: Yanyan

last: Wang

address: Chalmersplatsen 4

country: Sweden

institute: Chalmers University of Technology

summary: Saccharomyces cerevisiae has been used as a secretion host for production of various products, including pharmaceuticals. However, few antibody molecules have been functionally expressed in S. cerevisiae due to the incompatible surface glycosylation. Our laboratory previously isolated a group of yeast mutant strains with different α-amylase secretory capacities, and these evolved strains have showed advantages for production of some heterologous proteins. However, it is not known whether these secretory strains are generally suitable for pharmaceutical protein production. Here, three non-glycosylated antibody fragments with different configurations (Ran-Fab fragment Ranibizumab, Pex-the scFv peptide Pexelizumab, and Nan-a single V-type domain) were successfully expressed and secreted in three background strains with different secretory capacities, including HA (wild type), MA (evolved strain), and LA (evolved strain). However, the secretion of Ran and Nan were positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. Therefore, transcriptional analysis was performed to explore the fundamental changes triggered by the expression of the different pharmaceutical proteins in these selected yeast strains.

overall_design: To gain a sufficient comparative information, we designed a sample pool: four constructed strains LA.Nan, HA.Nan, LA.Pex and HA.Pex, each strain has biological triplicates. The expression of antibody fragments Nan and Pex has a opposite trend in the LA and HA strains. The production of Nan was higher in the HA.Nan strain in comparison to the LA.Nan strain. On the contrary, the production of Pex was lower in the HA.Pex strain than in the LA.Pex strain.

first: Xiaowei

last: Li

first: Xin

last: Chen

first: Jens

last: Nielsen

first: Dina

last: Petranovic

first: Verena

last: Siewers


title: The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice [Bisulfite-Seq]

city: Jiangsu Nanjig

first: Kezhi

last: Zheng

address: weigang NO.1

department: College of agriculture

institute: Nanjing Agricultural University

type: Methylation profiling by high throughput sequencing

summary: RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of Pol V, a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long non-coding RNAs. Pol V influences the accumulation of 24-nt siRNAs in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V-independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V-dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V-dependent loci but not on Pol V-independent loci. Our results reveal the function of rice Pol V for long non-coding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

overall_design: whole genome wide DNA methylation data by bisulfite sequencing

first: Dong-Lei

last: Yang


title: The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice [ncRNA-Seq]

overall_design: small RNA sequencing data


title: Suppression of plasmacytoid dendritic cell migration by compounds derived from natural medicines

city: Toyama

first: Takeshi

last: Yamamoto

address: 2630 Sugitani

institute: University of Toyama

summary: Background and Aims. Dendritic cells (DCs) play a pivotal role in maintaining immunological homeostasis by orchestrating innate and adaptive immune responses via migration to inflamed sites and the lymph nodes (LNs). Plasmacytoid DCs (pDCs) have been reported to accumulate in the colon of inflammatory bowel disease (IBD) patients and dextran sulfate sodium (DSS)-induced colitis mice. However, the role of pDCs in the progression of colonic inflammation remains unclear. Methods. 80 compounds in natural medicines were searched for inhibitors of pDC migration using bone marrow-derived pDCs (BMpDCs) and conventional DCs (BMcDCs). BALB/c mice were given 3% DSS in the drinking water for 7 days to induce acute colitis. Compounds, which specifically inhibited pDC migration, were administrated into DSS-induced colitis mice. Results. Astragaloside IV (As-IV) and oxymatrine (Oxy) suppressed BMpDC migration but not BMcDC migration. In DSS-induced colitis mice, the number of pDCs was markedly increased in the colonic lamina propria (LP), and the expression of CCL21 was obviously observed in colonic isolated lymphoid follicles (ILFs). As-IV and Oxy reduced symptoms of colitis and the accumulation of pDCs in colonic ILFs but not in the colonic LP. Moreover, in a BMpDC adoptive transfer model, BMpDC migration to colonic ILFs was significantly decreased by treatment with As-IV or Oxy. Conclusion. pDCs accumulated in the colon of DSS-induced colitis mice, and As-IV and Oxy ameliorated DSS-induced colitis by suppressing pDC migration to colonic ILFs. Accordingly, the selective inhibition of pDC migration may be a potential therapeutic approach for treating colonic inflammatory diseases.

overall_design: To elucidate the mechanism underlying inhibitory effects of Astragaloside IV and Oxymatrine on pDC migration, BMpDCs were generated from bone marrow cells isolated from the femurs and tibias of male BALB/c mice. Mature BMpDCs were treated with Astragaloside IV or Oxymatrine for 12 hours, followed by induction with CCL21 for 1 hour at 37°C.

first: Yue

first: Shusaku

last: Hayashi

first: Makoto

last: Kadowaki


title: Transcriptome profiles of tumors from 105K Tsc2-null cancer cells grown in C57BL/6J mice and treated with vehicle control, rapamycin and/or loratadine

city: Barcelona

first: Miguel Angel

last: Pujana

address: Gran Via 199

country: Spain

institute: Institut Catala d'Oncologia IDIBELL

summary: Characterization of the inhibitory effect of loratadine in monotherapy and combined with rapamycin on 105K Tsc2-null tumors.

overall_design: RNA sequencing of bulk mouse mammary tumors.

first: Roderic

last: Espin

first: Miquel


title: Transcriptional profiling of Pthlh-WT and PTHLH-KD pancreatic tumor cells

city: Worcester

first: Jason

last: Pitarresi

address: 364 Plantation Street

institute: University of Massachusetts Chan Medical School

summary: RNA-seq analysis of sorted Pthlh-WT vs Pthlh-KD in KPCY tumor cells

overall_design: Compared Pthlh-WT vs Pthlh-KD in two parental pancreatic tumor cell clones

first: Anil

last: Rustgi


title: An engineered IL-2 partial agonist promotes CD8+ T cell stemness

first: Peng

address: 9000 Rockville Pike

department: NHLBI

summary: Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients . Both the number of transferred T cells and their differentiation state are critical determinants of effective responses. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells and lower therapeutic efficacy, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in models of melanoma and acute lymphoblastic leukaemia. Thus, using engineered cytokine variants with distinctive properties is a promising strategy, with broad translational potential.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ChIP-Seq, RNA-Seq and ATAC-Seq analyses using TCR pre-activated CD8+ T cells that were expanded with either no cytokine, or with IL-2, H9 or H9T.

first: Fei

last: Mo

first: Zhiya

last: Yu

first: Jangsuk

last: Oh

first: Rosanne

last: Spolski

first: Liang

last: Zhao

first: Caleb

last: Glassman

first: Tori

middle: N

first: Yun

first: Filip

middle: M

last: Golebiowski

first: Dalton

last: Hermans

first: Sonia

last: Majri

first: Lora

last: Picton

first: Wei

last: Liao

first: Min

last: Ren

first: Xiaoxuan

last: Zhuang

first: Suman

last: Mitra

first: Jian-Xin

last: Lin

first: Luca

last: Gattinoni

first: Jonathan

middle: D

last: Powell

first: Nicholas

middle: P

last: Restifo

first: K. Christopher

last: Garica

first: Warren

last: Leonard


title: Inhibition of the neuromuscular acetylcholine receptor with atracurium activates FOXO/DAF-16-induced longevity (RNA-seq)

city: Amsterdam

first: Georges

middle: E.

last: Janssens

address: Meibergdreef 9

department: Laboratory Genetic Metabolic Diseases

institute: Amsterdam UMC

summary: Transcriptome-based drug screening is emerging as a powerful tool to identify geroprotective compounds to intervene in age-related disease. We hypothesized that, by mimicking the transcriptional signature of the highly conserved longevity intervention of FOXO3 (daf-16 in worms) overexpression, we could identify and repurpose compounds with similar downstream effects to increase longevity. Our in silico screen, utilizing the LINCS transcriptome database of genetic and compound interventions, identified several FDA-approved compounds that activate FOXO downstream targets in mammalian cells. These included the neuromuscular blocker atracurium, which also robustly extends both lifespan and healthspan in C. elegans. This longevity is dependent on both daf-16 signaling and inhibition of the neuromuscular acetylcholine receptor. Other neuromuscular blockers tubocurarine and pancuronium caused similar healthspan benefits. Together, these data demonstrate the capacity to mimic genetic lifespan interventions with drugs, and in doing so, reveal that the neuromuscular acetylcholine receptor regulates the highly conserved FOXO/DAF-16 longevity pathway.

overall_design: 4 samples each of C. elegans treated for 24 hours from larval stage 4 to day 1 of adulthood with 50 μM atracurium besylate or water vehicle controls

first: Rebecca

middle: L

last: McIntyre


title: Murine cytotoxic CD4 T cells

city: Philadelphia

first: Luis

last: Sigal

state: Pennsylvania

address: 233 S 10th St, BLSB709

institute: Thomas Jefferson University

summary: Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in anti-viral immunity.  For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection.  How viral infections induce CD4-CTL responses remains incompletely understood. Here we demonstrate that not only ECTV but also vaccinia virus and Lymphocytic Choriomeningitis virus induce CD4-CTL, but that the response to ECTV is stronger.  Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled.  We also show that Major Histocompatibility Complex Class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance.  Transcriptional analysis indicated that anti-viral CD4-CTL and non-cytolytic T Helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells.  Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment; and Runx3, required for CD8 T cell development and effector function.  However, at the protein level, CD4-CTL had higher levels of the three transcription factors suggesting that further post-transcriptional regulation is required for CD4-CTL differentiation.  Finally, using CRISPR-Cas9 deletion of Runx3 in CD4 T cells, we demonstrate that the development of CD4-CTL but not of classical Th1 CD4 T cells requires Runx3 following ECTV infection.  These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of post-transcriptionally regulated Runx3 in this process.

overall_design: Murine T cell populations from the liver were sorted on a flow cytometer and total RNA was extracted at 7 days following ectromelia virus infection.  Granzyme B (Gzmb) reporter mice (C57BL/6-Gzmbtm1Asmv/Orl) were used to identify cytotoxic T cells.  CD44 low Gzmb-, CD44+ Gzmb-, and CD44+ Gzmb+ CD4 T cells as well as CD44+ Gzmb+ CD8 T cells were sorted from a total of 10 livers from ectromelia virus infected mice.  As controls, CD4 and CD8 T cells that were CD44 low and CD44+ were also sorted from spleens of naive reporter mice.  Total RNA was extracted for up to 10E6 cells for each population using RNA Clean and Concentrator-5' kit from Zymo Research into 25 microliters elution buffer, and 3 samples for each population were collected across independent experiments (total of 24 samples).  RNA was sent to Novogene for RNA sequencing with 20 million, 150 PE reads and bioinformatic analysis.

first: Cory

last: Knudson


title: RNA-sequencing data from PLC/PRF/5 cells with NC or USP22 knockdown (shUSP22) under hypoxic condition

status: Public on Jul 05 2021

city: Hangzhou

first: Sunbin

last: Ling

state: Zhejiang

address: 79 Qingchun Road

institute: Zhejiang University

summary: We use RNA-sequencing to evaluate the effect of USP22 knockdown on the gene changes in HCC cells

overall_design: examing two replicates of each group under hypoxic condition


title: Compartment-specific regulation of gene expression orchestrates homeostatic synaptic scaling

city: Zurich

first: Gerhard

last: Schratt

address: Winterthurerstrasse 190

department: HEST

country: Switzerland

institute: ETH Zürich

summary: Synaptic scaling is a form of homeostatic plasticity which allows neurons to reduce their action potential firing rate in response to chronic alterations in neural activity. Synaptic scaling requires profound changes in gene expression, but the relative contribution of local and cell-wide mechanisms to synaptic scaling is controversial.  Here we performed a comprehensive multi-omics characterization of the somatic and process compartments of primary rat hippocampal neurons during synaptic scaling. Thereby, we uncovered highly compartment-specific and correlated changes in the neuronal transcriptome and proteome. Specifically, we identified highly compartment-specific downregulation of crucial regulators of neuronal excitability and excitatory synapse structure. Motif analysis further suggests an important role for trans-acting post-transcriptional regulators, including RNA-binding proteins and microRNAs, in the local regulation of the corresponding mRNAs. Altogether, our study indicates that compartmentalized gene expression changes are widespread in synaptic scaling and might co-exist with neuron-wide mechanism to allow synaptic computation and homeostasis.

overall_design: Investigation of a compartment specific ptx effect on rat hippocampal neurons

first: David

last: Colameo

first: Marek

last: Rajman

first: Michael

last: Soutschek


title: Transcriptomic analysis of patient-derived ex vivo models of pancreatic cancer

status: Public on Jul 03 2021

city: London

last: Osuna de la Peña

address: 5 University Street

department: Respiratory Medicine

institute: University College London

summary: The aim of this study is to compare the transcriptomic profiles of various ex vivo models of pancreatic cancer. First, primary tumours and their corresponding xenografts in nude mice were analysed by RNA-seq. Monocultures of pancreatic cancer cells derived from the xenografts were then prepared as 2D monolayers, Matrigel-embedded organoids, spheres in suspension and 3D cultures in self-assembling peptide amphiphile (PA) hydrogels. Seven-day cultures were then analysed by RNA-seq. The results suggest that all ex vivo monocultures retain patient-specific transcriptional profiles, especially cancer stem cell signatures, while being deficient in the expression of stromal components such as the core matrisome. Correlations with the primary tumours were generally higher in PA hydrogels than organoids. Biased gene expression signatures were identified in certain models. This is the first study to explore the transcriptomic signatures of four different ex vivo models matched to their primary tumours of origin.

overall_design: 29 patient-matched pancreatic adenocarcinoma (PDAC) samples were analysed: three primary PDAC tumour samples, six PDAC monocultures in PA (3 patients, duplicates), six PDAC organoid monocultures in Matrigel (3 patients, duplicates), six PDAC monocultures in 2D (3 patients, duplicates), six PDAC monocultures in suspension (3 patients, duplicates), and two primary patient-derived xenografts in grown in nude mice.

first: Daniela

last: Loessner


title: Cytomegalovirus-mediated expansion of IL-15-responsive innate-memory cells with SIV killing function

status: Public on Jul 02 2021

city: Sacramento

first: Dennis

middle: J.

last: Hartigan-O'Connor

state: CALIFORNIA

address: 3146 One Shields Avenue, Tupper Hall

department: Medical Microbiology and Immunology

institute: University of California, Davis

summary: We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells—and marked expansion of innate-memory CD8+ T cells. These cells express high levels of NKG2A/C and the IL-2- and IL-15-receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C+CD122+CD8+ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I-deficient K562 targets and prompt IFN-ɣ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1-vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C+CD8+ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.

overall_design: 6 RhCMV positive animals and 6 RhCMV negative animals were included in this study.

first: Gema

last: Méndez-Lagares

first: Ning

last: Chin


title: The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice

first: Kezhi

last: Zheng

summary:
RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of Pol V, a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long non-coding RNAs. Pol V influences the accumulation of 24-nt siRNAs in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V-independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V-dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V-dependent loci but not on Pol V-independent loci. Our results reveal the function of rice Pol V for long non-coding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

This SuperSeries is composed of the SubSeries listed below.

overall_design:
Refer to individual Series

fem3-2_seedling_sRNA
pol iv-1_seedling_sRNA
fem3-1_seedling_sRNA
fem3-2_seedling_Bisulfite-Seq
TP309_seedling_Bisulfite-Seq
GAS_seedling_Bisulfite-Seq
pol iv-1_seedling_Bisulfite-Seq
fem3-1_seedling_Bisulfite-Seq


title: Oct4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human [RNA-seq}

city: Saint Louis

first: Bo

last: Zhang

state: Missouri

address: 4515 McKinley Research Bldg 03212

department: Developmental Biology

institute: Washington University School of Medicine

summary: Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their physical interactome in human ESCs (hESCs) has not been explored thus far. Here we mapped the protein-protein interactions of OCT4 in naive and primed hESCs, revealing extensive connections to ATP-dependent nucleosome remodeling complexes. In naive hESCs, OCT4 is associated with both BRG1 and BRM, the two core ATPases of the BAF complex. Genome-wide location analyses and genetic deletion studies reveal that these two enzymes exert a functionally redundant role in transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs OCT4 cooperates with BRG1 and the transcription factor SOX2 to create an open chromatin architecture at neural lineage associted genes. This work offers insight into the regulation of human stem cell identity and reveals how a common transcription factor utilizes differential BAF complex composition to control distinct transcriptional programs in naive and primed hESCs.

overall_design: To identify the functional roles of the core BAF ATPases, BRG1 and BRM, in human pluripotency, we performed RNA-seq analysis on BRG1-KO and BRM-KO naïve hESCs and BRG1-KO cells during the naive-to-primed transition.

first: Thorold

last: Theunissen

first: Huang

last: Xin

first: Park

last: Kyoung-mi

first: Paul

last: Gontarz


title: Oct4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Oct4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human [CUT&Tag]

overall_design: CUT&Tag analysis was performed in primed and naïve hESCs to map genome-wide KLF4 binding sites.

first: Chen

last: Dong


title: Oct4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human [ATAC-seq]

overall_design: ATAC-seq analysis was performed to map the consequences of BRG1 removal on global chromatin accessibility in naïve hESCs and during the naïve-to-primed transition.

first: Ting

last: Wang

first: Xiaoyun

last: Xing


title: Meiotic recombination mirrors patterns of germline replication in mice and humans

city: Bethesda

first: Kevin

last: Brick

address: 5/205 Memorial Drive

department: GBB

institute: NIDDK

summary: Genetic recombination generates novel trait combinations, and understanding how recombination is distributed across the genome is key to modern genetics. The PRDM9 protein defines recombination hotspots; however, megabase-scale recombination patterning is independent of PRDM9. The single round of DNA replication, which precedes recombination in meiosis, may establish these patterns; therefore, we devised an approach to study meiotic replication that includes robust and sensitive mapping of replication origins. We find that meiotic DNA replication is distinct; reduced origin firing slows replication in meiosis, and a distinctive replication pattern in human males underlies the subtelomeric increase in recombination. We detected a robust correlation between replication and both contemporary and historical recombination and found that replication origin density coupled with chromosome size determines the recombination potential of individual chromosomes. Our findings and methods have implications for understanding the mechanisms underlying DNA replication, genetic recombination, and the landscape of mammalian germline variation.

overall_design: We devised a tripartite method to study DNA replication in mammalian meiosis. We mapped origins of replication in mouse testis, inferred replication timing from whole genome sequencing of meiotic S-phase nuclei, and used in-silico modelling to infer the parameters of meiotic replication relative to other cell types.

first: Florencia

last: Pratto

first: Geng

last: Cheng

first: Kwan-Wood

middle: G

last: Lam

first: Daisy

last: Dahiya

first: Jeff

last: Cloutier

first: Stephen

last: Wellard

first: Phil

last: Jordan

first: Rafael

last: Camerini-Otero


title: Genome-Wide Profiling of DNA Methylation and Gene Expression induced by Alpha-Synuclein in Murine Microglia [RNA-seq]

city: Grand Forks

first: Junguk

last: Hur

state: ND

address: 1301 N. Columbia Rd. Stop 9037

department: Biomedical Sciences

institute: University of North Dakota

summary: BACKGROUND: Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. However, the influence α-syn has on DNA methylation and gene expression patterns for other genes has not been thoroughly explored. Our objective is to determine gene expression and methylation changes in microglia induced by aggregate α-syn.METHODS: Transgenic mThy1-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-induced changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data.RESULTS: Microglial DNA isolated from our 3-month cohort had 5,315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3,742 DMGs and 3,766 DEGs in 13-month mThy1-Asyn mice.These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months.CONCLUSION: We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.

overall_design: We performed transcriptomic and methylomic analysis of murince brain homogenate subjected to microglia selection enrichment from mice overexpressing full-length human wild-type α-syn under the murine Thy-1 promoter on the X chromosome. Transcriptomic and methylomic profiles were analyzed for biological meaning using Reactome pathway database, Gene ontology, and Kyoto Encyclopedia of Genes and Genomes on differentially expressed genes (DEGs) and differentially methylated genes (DMGs). Pathways were also examined by enrichment network anlaysis and MCODE to identify clusters of highly connected pathways. HEM samples are mice overexpressing human alpha-synuclein while WT mice are  controls. The corresponding background of these mice is the B6D2F1/J mice (stock# 100006) from The Jackson Laboratory (Bar Harbor, ME). There are eight 13 month old HEM mice and four 13 month wild-type controls. There are four 3 month HEM mice and three 3month wild-type controls.

first: James

last: Porter

first: Brett

last: McGregor

first: Othman

last: Ghribi

first: Kai

last: Guo

first: Jared

last: Schommer

first: M

middle: O

last: Raihan


title: Gene expression of lung tissues in non-human primate (NHP, rhesus macaque) after exposure to thoracic irradiation

city: Baltimore

first: Wanchang

last: Cui

state: Maryland

address: 10 South Pine St

department: Radiation Oncology

institute: University of Maryland Baltimore

summary: Radiation-induced lung injury is a serious health threat to victims of radiological incidents or patients receive radiation in the thorax. Studying the gene expression in lung tissues after radiation exposure helps to understand the mechanism of radiation-induced lung injury.

overall_design: Rhesus macaques were exposed to 9.0 to 12.0 Gy whole thoracic lung irradiation (WTLI) and followed for 180 days. Each animal was euthanized when the pre-determined euthanasia criteria were met or past the 180 days. Lung tissues from the euthanized animals were used for gene expression profiling analysis. n=14 in the decedent group, n=12 in the survivor group, and n=5 in the non-irradiated group.

first: Thomas

last: MacVittie


title: RNA sequencing of adipose tissue-derived stromal cells (ADSCs) with or without Sal-B addition

status: Public on Jul 01 2021

city: Shanghai

first: Sun

middle: Jia

last: Ming

address: No. 639, Zhi Zao Ju Road,

department: Department of Plastic & Reconstructive Surgery

institute: Shanghai Ninth People’s Hospital

summary: Salvianolic acid B (Sal-B) is the most abundant and bioactive water-soluble compound in Salvia miltiorrhiza. Studies have shown that salvianolic acid B increases the mRNA expression of adipogenic transcription factors, including PPARγ, C/EBPα and PPARα, in 3T3-L1 preadipocytes to increase glucose uptake and mitochondrial respiration, reduce glycerol release and promote adipocyte differentiation. However, the effect of Sal-B on graft fat survival and its specific mechanism have not been investigated. So we dicided using h-ADSCs to see its transcriptomes changing after Sal-B addition.

overall_design: Examination of h-ADSCs RNA sequencing comparing the Sal-B addition(50μmol/L, four days and eight days) group and control group

first: Jiaming

last: Sun

first: Li

last: Yu


title: FOXO1 mediates hypoxia-induced G0/G1 arrest in ovarian somatic granulosa cells via activating the TP53INP1-p53-CDKN1A pathway

city: Nanjing

first: Chengyu

last: Li

address: College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing, 210095, China.

summary: The development of ovarian follicles constitutes the foundation of female reproduction. The proliferation of granulosa cells (GCs) is a basic process required to ensure normal follicular development. However, the mechanisms involved in controlling GC cell cycle are not fully understood. Here, by performing gene expression profiling, we showed that cell cycle arrest at G0/G1 phase is highly correlated with pathways associated with hypoxic stress and FOXO signalling. Specifically, the elevated proportion of GCs at the arrested G0/G1 phase was accompanied by increased nuclear translocation of FOXO1 under conditions of hypoxia both in vivo and in vitro. Actually, phosphorylation of 14-3-3 by the JNK kinase is required for hypoxia-mediated FOXO1 activation and the resultant G0/G1 arrest. Notably, FOXO1 mutant without DNA-binding activity failed to induce G0/G1 arrest of GCs during hypoxia. Importantly, we identified a new target gene of FOXO1, namely TP53INP1, which contributed to the suppression of the G1-S cell cycle transition in response to hypoxia. Furthermore, we demonstrated that the inhibitory effect of the FOXO1-TP53INP1 axis on GC cell cycle is mediated through a p53-CDKN1A-dependent mechanism. These findings might provide avenues for the clinical treatment of human infertility caused by impaired follicular development.

overall_design: Granulosa cells (GCs) in porcine antral follicles were isolated for flow cytometric detection of cell cycle. Based on the ratio of cells in G0/G1 phase, the remaining GCs in each follicles were classified into 4 groups with 2 high G0/G1 distribution groups (high groups) and 2 low G0/G1 distribution groups (low groups), both of which were processed for RNA-seq analysis.

first: Ming

last: Shen

first: Honglin

last: Liu


title: Genome-Wide Profiling of DNA Methylation and Gene Expression induced by Alpha-Synuclein in Murine Microglia


title: Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer [RNA-seq]

city: Little Rock

first: Allen

last: Gies

state: AR

address: 4301 W. Markham

department: Biochemistry and Molecular Biology

institute: University of Arkansas for Medical Sciences

summary: Triple negative breast cancer is an aggressive type of breast cancer with very little treatment options. TNBC is very heterogeneous with large alterations in the genomic, transcriptomic, and proteomic landscapes leading to various subtypes with differing responses to therapeutic treatments. We applied a multi-omics data integration method to evaluate the correlation of important regulatory features in TNBC BRCA1 wild-type MDA-MB-231 and TNBC BRCA1 5382insC mutated HCC1937 cells compared with normal epithelial breast MCF10A cells. The data includes DNA methylation, RNAseq, protein, phosphoproteomics, and histone post-translational modification. Data integration methods identified regulatory features from each omics method had greater than 80% positive correlation within each TNBC subtype. Key regulatory features at each omics level were identified distinguishing the three cell lines and were involved in important cancer related pathways such as TGFbeta signaling, PI3K/AKT/mTOR, and Wnt/beta-catenin signaling.

overall_design: RNA-seq and EPIC Bead Chip

first: Kevin

last: Chappell

first: Kanishka

last: Manna

first: Charity

last: Washam

first: Stefan

last: Graw

first: Duah

last: Alkam

first: Matthew

last: Thompson

first: Maroof

middle: K

last: Zafar

first: Lindsey

last: Hazeslip

first: Christopher

last: Randolph

first: Jordan

last: Bird

first: Alicia

last: Byrd

first: Sayem

last: Miah

first: Stephanie

last: Byrum


title: Comparative analysis of gene expression after Oct4 or Scrambled siRNA knockdown in HNSCC cell lines

city: Dresden

first: Vasyl

last: Lukiyanchuk

address: Handelallee 26

institute: OncoRay

summary: By employing a global gene expression profiling, we performed analysis of the molecular pathways which are deregulated in  head-and-neck cancer cell lines FaDu, Cal33 and UTSCC5 after siRNA mediated knockdown of Oct4 comparing to Scrambled.

overall_design: Cells transfected with Scrambled or Oct4 siRNA were compared. The experiment was done in 3  head-and-neck cancer cell lines and repeated 3 times.

first: Anna

last: Dubrovska

first: Linda

last: Hein


title: IL-1-driven stromal-neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies

city: Oxford

first: Fiona

last: Powrie

address: Roosevelt Drive

department: Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences

institute: University of Oxford


title: Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer

first: Allen

last: Gies


title: Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer [methylation]

summary:
Triple negative breast cancer is an aggressive type of breast cancer with very little treatment options. TNBC is very heterogeneous with large alterations in the genomic, transcriptomic, and proteomic landscapes leading to various subtypes with differing responses to therapeutic treatments. We applied a multi-omics data integration method to evaluate the correlation of important regulatory features in TNBC BRCA1 wild-type MDA-MB-231 and TNBC BRCA1 5382insC mutated HCC1937 cells compared with normal epithelial breast MCF10A cells. The data includes DNA methylation, RNAseq, protein, phosphoproteomics, and histone post-translational modification. Data integration methods identified regulatory features from each omics method had greater than 80% positive correlation within each TNBC subtype. Key regulatory features at each omics level were identified distinguishing the three cell lines and were involved in important cancer related pathways such as TGFbeta signaling, PI3K/AKT/mTOR, and Wnt/beta-catenin signaling.
EPIC Bead Chip

overall_design: Kevin, Chappell

first: Kanishka

last: Manna


title: 14-kDa Phosphohistidine Phosphatase is a Potential Therapeutic Target for Liver Fibrosis

city: Beijing

first: Anjian

last: Xu

address: No. 95 Yong'an road, Xicheng District, Beijing

institute: Capital medical university

summary: Liver fibrosis, a major cause of morbidity and mortality worldwide, leads to liver damage, seriously threatening human health. In our previous study, we demonstrated that 14-kDa phosphohistidine phosphatase (PHP14) was upregulated in fibrotic liver tissue and involved in the migration and lamellipodia formation of hepatic stellate cells (HSCs). In this study, we evaluated PHP14 as a therapeutic target for liver fibrosis and investigated the mechanism by which it mediates liver fibrosis. AAV-Phpt1 administration significantly attenuates CCl4-induced liver fibrosis in mice. In particular, fibrosis-associated inflammatory infiltration was significantly suppressed after PHP14 knockdown. Mechanistically, PHP14 regulated macrophage recruitment, infiltration and migration by affecting podosome formation of macrophages. Inhibition of PHP14 decreased the expression of the fibrogenic signature at the early stage of liver fibrogenesis and the activation of HSCs in vivo. Thus, PHP14 can be considered a potential therapeutic target for liver fibrosis.

overall_design: liver mRNA profiles of normal, CCl4-treated adeno-associated virus (AAV)-Ctrl and AAV-shPhpt1 mice after 1 week of CCl4 injection


title: RNA sequencing of FACS-sorted cell populations from inflamed and non-inflamed endoscopic biopsies of patients with IBD, or from blood of patients with IBD and healthy donors

summary: biopsies from the most inflamed area were sequenced where applicable. FACS-sorted cell populations: N=neutrophils; M=mononuclear phagocytes; S=stromal cells; E=eosinophils

overall_design: SmartSeq2 RNA-sequencing, transcript quantification

first: Kevin

last: Rue-Albrecht

first: Matthias

last: Friedrich

first: Mathilde

last: Pohin


title: RNA-sequencing of whole tissue samples from patients with inflammatory bowel disease and colorectal cancer

summary: 3'-end RNA sequencing of inflamed and non-inflamed resected intestial tissue from IBD patients, as well as from non-tumor intestinal tissue resected from patients with colorectal cancer

overall_design: transcript quantification in inflamed, non-inflamed and control tissue

first: Samuel

last: Bullers

first: Zoe

last: Christoforidou


title: RNA-sequencing of cytokine-stimulated cultured human fibroblasts

summary: Smart-Seq2 RNA sequencing of Ccd18-Co human intestinal fibroblasts, unstimulated or stimulated with cytokines

overall_design: transcript quantification in Ccd18-Co cells, unstimulated or pre-stimulated for 3 hours with 100ng/ml TNF-alpha, or 0.01ng/ml IL-1beta (n=6 each)